Effectiveness of repeated mutagenesis of sesame crosses for enhancing polygenic variability in F2M2 generation

The value of combining hybridization and mutagenesis in sesame was examined to determine if treating hybrid sesame plant material with mutagens generated greater genetic variability in four key productivity traits than either the separate hybridization or mutation of plant material. In a randomized block design with three replications, six F2M2 varieties, three F2varieties, and three parental varieties were assessed at Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India. The plant characteristics height, number of seed capsules per plant, and seed yield per plant had greater variability in the F2M2 generation than their respective controls (F2), however, the number of primary branches per plant varied less than in the control population. The chances for trait selection to be operative were high for all the characteristics examined except the number of primary branches per plant, as indicated by heritability estimates. Increases in the mean and variability of the characteristics examined indicted a greater incidence of beneficial mutations and the breakdown of undesirable linkages with increased recombination. At both phenotypic and genotypic levels strong positive correlations between both primary branch number and capsule number with seed yield suggest that these traits are important for indirect improvement in sesame seed yield. As a result of the association analysis, sesame seed yield and its component traits improved significantly, which may be attributed to the independent polygenic mutations and enlarged recombination of the polygenes controlling the examined characteristics. Compared to the corresponding control treatment or to one cycle of mutagenic treatment, two cycles of mutagenic treatment resulted in increased variability, higher transgressive segregates, PTS mean and average transgression for sesame seed yield. These findings highlight the value of implementing two EMS treatment cycles to generate improved sesame lines. Furthermore, the extra variability created through hybridization may have potential in subsequent breeding research and improved seed yield segregants may be further advanced to develop ever-superior sesame varieties.


Introduction
Sesame (Sesamum indicum L.) is primarily produced commercially for its edible seed, oil, and flavour. Sesame seeds contain 40-58% oil, 20-25% protein, and 13.5% carbohydrate [1]. A 100 g sample of sesame seeds contains the following nutrients: 49.7 g of fat, 9.85 g of carbohydrates, 17.6 g of protein, 14.9 g of fibre, and approximately 4.48 g of ash [2]. Sesame seeds also include sesamolin and sesamin which are natural preservatives of sesame food products: sesamin is an antioxidant with anti-inflammatory properties [3]. As well, sesame contains significant amounts of high-quality protein with a balanced amino acid profile [4]. Sesame has not been the subject of much research by national or international agricultural research organizations, and as a result research into this crop has lagged significantly behind other important oil seed crops.
Sesame is grown widely in several countries, including China, India, Ethiopia, Uganda, Nigeria, and Myanmar. In India the crop is grown on 1.52 million hectares, with a production of 0.66 million metric tons and productivity of 433 kg/ha, compared to the global average of 512 kg/ha [5]. There is potential to improve from the existing low yield productivity in India.
The commercial production of sesame is constrained by a lack of improved, modern cultivars; existing varieties are susceptible to disease, establish poorly, have unpredictable growth, ripen unevenly, and are at risk of capsule shattering and profuse branching which result in low harvest indices [6]. Sesame improvement has been explored using a variety of breeding techniques; however, these have only resulted in minor increases in productivity. The natural genetic variability of sesame has been depleted by extensive inbreeding types over a long period of time [7].
Recombination and mutation breeding have been identified as pathways by which genetic variability may be increased to facilitate the selection of outstanding genotypes: the effectiveness of these techniques has previously been demonstrated in other crop plants. Mutation breeding is tool for rapidly generating high quality outcomes relatively quickly. Nonetheless, due to their narrow genetic base, the mutation in homozygous genotypes has had limited success. When subjected to mutagenesis, the homozygous genetic material rarely yields favourable mutants due to low mutability. To find such economic mutants in the offspring, it is necessary to evaluate large populations using effective screening procedures. Recombination breeding is a popular technique for crop improvement that produces new and enhanced recombinants by combining hybridization among parents, followed by selection in subsequent generations. This makes it easier to combine advantageous traits into one genotype. However the potential of recombination in sesame is constrained by correlations between favourable and negative traits: the ability to identify improved genotypes is reduced because the necessary amount of variability between traits does not exist.
Recombination in combination with mutant breeding has been proposed as an innovative strategy to overcome the constraints of traditional breeding practices in sesame. The heterozygosity of the hybrids provides the mutagen a wide genetic foundation to work on, generating variability and facilitating additional selection. Mutagenesis of the hybrid species induces additional micro-mutations [8], enhances recombination of different traits [9], breaks undesirable linkages [10], and creates greater variability than the sum of the variances of mutagenized genotypes and segregated hybrid progenies separately [11,12]. The potential for enhanced variability has been demonstrated following hybrid mutagenic treatments and improved cultivars have been produced in several crops using this technique. However, research on this approach in sesame is limited.
This paper reports on an analysis conducted to evaluate the outcome in sesame of hybridization with both non-recurrent and recurrent mutagenic treatments on the variability of important quantitative traits related to yield during the F 2 M 2 generation. Three inter-varietal crosses were made between three adapted sesame varieties (Nirmala, Prachi, and Amrit). These research findings provide insights into the usefulness of hybridization alone and hybridization combined with recurrent and non-recurrent EMS mutagenesis on the potential to increase sesame yield. The potential to identify any additional variability created through hybridization would inform future sesame breeding activities and the selection of superior lines that, after rigorous testing, may be developed into releasable varieties.

Selection of sesame parent varieties
Three released varieties of sesame, Nirmala, Prachi, and Amrit, which are recommended for the state of Odisha in eastern India were used as potential parent varieties for this research. Important characteristics of these cultivars are outlined in Table 1.

Experimental location
A field study was conducted at the Economic Botany-II, Genetics and Plant Breeding Department, College of Agriculture, OUAT Bhubaneswar, India.

Sesame hybridization and cross development
To generate different cross combinations (Nirmala x Prachi, Nirmala x Amrit, and Prachi x Amrit), the parental varieties were grown at staggered intervals of seven days. Each crop was grown using agronomic practices recommended within Odisha. To achieve the cross combinations when the corolla of the intended male parent was about to open the plant was emasculated by removing the flower's epipetalous corolla and applying a speck of fevicol to it [13]. Fevicol was also sprayed on the flower's top to prevent the corolla from opening any further. All of these processes were done in the late afternoon. Among the three varieties, three crosses were achieved, excluding reciprocals.

Chemical mutagen selection
Mutations in genes are a common result of chemical mutagens [14,15]. The most potent and effective chemical mutagen is ethyl methane sulfonate (EMS) [16]. Previous research has demonstrated the usefulness of EMS to induce beneficial mutations in sesame, and in particular that lower concentrations of EMS have the highest mutagenic efficiency and effectiveness in inducing beneficial mutations [17][18][19][20][21][22][23]. Accordingly, we selected a dosage rate of 0.5% EMS (for three hours per cycle) for the treatment of sesame seed materials in this research.

Sesame field experiment
The produced F 1 hybrids were subjected to two cycles of ethyl methane sulfonate treatment, at a dose of 0.5% per cycle for three hours. The number of treated and crossed populations (excluding parents) increased from 6 in the F 1 M 1 to 9 in the F 2 M 2 generation, when the generations were advanced from F 1 M 1 to F 2 M 2 with and without recurring chemical mutagenesis (Fig 1). The nine populations that made up the material for the F 1 M 1 generation included the untreated F 1s , the parental varieties, and the EMS-treated F 1s (classified as F 1 1 M 1s ) from the crosses Nirmala x Prachi, Nirmala x Amrit, and Prachi x Amrit. Using bulk seeds from the sample plants, the materials from the F 1 M 1 generation were advanced to the F 2 M 2 .
The treated populations, i.e., F 1 M 1s , were advanced to the next generation with and without recurrent mutagenic treatment. Three additional populations arose from two cycles of EMS treatment, and the remaining six populations were the same as in F 1 M 1 except for the advancement of the generation to F 2 M 2 and F 2 . Therefore, in the F 2 M 2 generation, there were 12  Table 2.
To grow plants of the six F 2 M 2s , three F 2s , and three parental sesame types, a randomized complete block design with three replications was used. Sesame seed was sown in plots of 8 rows, each 2.5 meters long, with 30 cm x 10 cm spacing. After dibbing two to three seeds per hill, subsequent thinning left only one seedling per hill for a good crop stand. Other crop management (fertilizer application, watering, and other cultivation activities) was undertaken following the standard recommended practice for sesame cultivation in Odisha recommended by Odisha State Government.

Data sampling
Thirty plants were randomly selected from each treatment plot. From each plant height, number of primary branches per plant, the number of capsules per plant, and yield were all recorded.

Statistical evaluation
Using the means of each of the four measured characteristics (plant height, number of primary branches, number of capsules per plant, and yield). The performance of each of the sesame lines was analysed statistically. The standard deviation and coefficient of variation of each of the relevant traits were calculated separately for each treatment using an analysis of variance (ANOVA), and F and t-tests were used to assess the significance of differences between treatments [24,25]. Using standard formulae, the heritability (broad sense) coefficients of various traits were calculated [26,27].
To investigate the nature of the correlation between the contributory characteristics, all potential correlations among the four characteristics were calculated for each treatment/population at both phenotypic and genotypic levels. To evaluate whether a correlation coefficient was statistically significant, a t-test at (n-2) degrees of freedom was performed [28]. The frequency and magnitude of positive transgressive segregation for seed yield per plant were examined at the level of the individual plant. In the individual plant analysis mutants were compared to the highest-yielding individual of its better parent. By deducting the mean of positive transgressive segregates (PTS mean) from the better parent mean, the extent of transgression was determined [29]. Table 3 shows the results of an ANOVA of means and heritability estimates for four key characteristics (plant height, the number of primary branches per plant, number of capsules per plant and the seed yield per plant) in the F 2 M 2 sesame generation. There are significant differences in means among treatments for all characteristics except for the number of primary branches per sesame plant.

Variability and heredity in the F 2 M 2 sesame generation
The significant heritability estimates for the different characteristics ranged from 55.70% for the sesame seed yield per plant to 81.30% for the number of capsules per plant. The heritability estimates were high for the number of capsules per plant; moderate for plant height and seed yield per plant, and low (and without significant difference) for the number of primary branches per plant. In Tables 4 and 5, an analysis of the variance of the standard deviation and the coefficient of variation of the different traits is shown.
The F test indicated significant variations between treatments in the standard deviation and coefficient of variability for all the examined characteristics, except the number of primary branches per plant. Considerable variation in these parameters among genotypes was expected because the parent materials involved were genotypically homogenous and segregating populations such as the F 2s and F 2 M 2s . Tables 6 to 9 show the variability parameters of all the characteristics examined in the F 2 M 2 sesame generation. Plant height variation was reduced in all mutants in F 2 1 M 2 and F 2 2 M 2 ( To ascertain which recurrent EMS treatment cycle of hybrids would be most beneficial to enlarge variability in subsequent generations, changes in the mean and standard deviation (expressed as percentages of the corresponding control) of several traits were also studied. Two cycles of mutagenesis treatment were applied to the hybrid materials, starting with F 1s . Hybrid plant height mean as a percentage of the control plant height mean decreased with increasing number of EMS treatments for up to two mutagenic cycles in the crosses Nirmala x Amrit and Prachi x Amrit, and increased from one to two mutagenic cycles in the cross Nirmala x Prachi (Fig 2a). For different F 4 M 4 populations of the cross Nirmala x Prachi trends from one to two mutagenic cycles showed little change in mean plant height as a percentage of the control plant height. In the remaining mutant crosses s of Nirmala x Amrit and Prachi x Amrit, the mean standard deviation (as a percentage of the control plant standard deviation) decreased with increasing number of mutagenic cycles in the F 2 M 2 populations (Fig 2b). Table 7 illustrates characteristics of the number of primary branches per plant in the F 2 M 2 generation. The 1 F 2 , 3   control treatment increased slightly with increasing EMS treatment across all F 2 M 2s populations. Table 8 shows characteristics of the number of capsules per plant, which was highest (30.14-136.94) in the 3 F 2 2 M 2 population and lowest (35.68-111.28) in the 2 F 2 population. M 2s populations had similar means, than their respective controls (F 2s ), and all mutants showed a significantly higher standard deviation (F 2s ). The F 2 2 M 2s population (two cycles mutated populations) had a higher standard deviation in the number of capsules per plant than the F 2 1 M 2s population (one cycle EMS treated populations), indicating that two cycles of EMS treatment may generate more variability than the corresponding controls or the one cycle of EMS treatment for this trait. Both the mean and standard deviation (as a percentage of the relevant control) of the number of capsules per plant increased from one to two cycles of mutagenesis (Fig 4a & 4b).

Comparison of characteristics in the F 2 M 2 sesame generation
In  The progenies of the F 2 M 2 (Nirmala x Prachi) cross which were subjected to one and two cycles of EMS had significantly greater seed yield standard deviations than the corresponding controls. In comparison to the corresponding one-cycle mutant populations, the two-cycle mutated progenies, 1 F 2 2 M 2 and 2 F 2 2 M 2 , had a greater standard deviation in seed yield, while the standard deviation was smaller in the 3 F 2 1 M 2 and 3 F 2 2 M 2 populations compared to the F 2 (Prachi x Amrit) control. The 2 F 2 1 M 2 population had a seed yield standard deviation comparable to that of the control F 2 (Nirmala x Amrit), however, the 2 F 2 2 M 2 population had a significantly higher standard deviation. Mean seed yield values (as a percentage of the respective control) increased between one to two cycles of mutagenic treatment in the Nirmala x Amrit cross, while they decreased between one to two cycles of mutagenic treatment in the Nirmala x Prachi and Prachi x Amrit crosses (Fig 5a). One to two cycles of mutagenic treatment resulted in an increase in variability for mean seed yield, but the amount of variability varied for different cross combinations, as shown in Fig 5b.

Correlatingsesame yield and its constituent traits in the F 2 M 2 generation
Phenotypic correlation in populations of different genotypes consists of both genotypic and environmental components. In contrast, the association between traits in populations with genotypically uniform individuals, such as pure-line varieties, is entirely due to environmental factors. A genotypic correlation is the outcome of genetic association, while an environmental correlation is the consequence of environmental factors which can influence how characteristics manifest. The nature and strength of the relationship between characteristics alters with alterations in the genotypic makeup of the population, and these changes depend on the type and extent of genetic alterations. In particular, in terms of the extent of any genetic change the relationship between characteristics may dissipate completely or partially if the associated characteristics' genetic changes are independent. Tables 10 and 11 Table 12 shows the frequency and magnitude of positive transgressive variants for sesame seed yield per plant in the F 2s and F 2 M 2s populations. All the F 2 M 2 and 3 F 2 populations generated   M 2 ) showed a greater range of variation relative to their respective controls (F 2 ) ( Table 9). When compared to their respective control populations 3 F 2 , the 3 F 2 1 M 2 and 3 F 2 2 M 2 populations had a lower range of variation. Of the six F 2 M 2s populations achieved subsequent to one or two cycles of EMS treatment, two populations had decreased means, one population had a similar mean, and three populations had increased means relative to their respective control (F 2 ) populations. Similarly, out of six F 2 M 2 s, one had a decreased mean, one had a similar mean, and four had an increased mean for seed yield per plant compared to the respective F 2 populations. The one-and two-cycle EMS-treated populations of F 2 M 2 (Nirmala x Prachi) and F 2 M 2 (Nirmala x Amrit) had significantly greater variability than their respective controls. In general, the two-cycle EMS-treated populations 1 F 2 2 M 2 and 2 F 2 2 M 2 had greater variability in seed yield per plant than the respective one-cycle EMS-treated populations. Recurrently mutagenized populations had greater variability than corresponding one-cycle EMS-treated populations in terms of seed yield per sesame plant.

Transgressive segregation at the level of individual plants in the F 2 M 2 sesame generation
Overall, F 2 M 2 populations resulted in a negative shift in mean plant height, and a positive shift in mean capsule number and seed yield in mutants compared to the respective control populations. Similar results have been noted elsewhere for plant height and seed yield [34], for the number of capsules per plant and seed yield per plant [35], for capsule number [36], for seed yield [37], and for capsule number and seed yield [38].
The analysis of the F 2 M 2 populations indicated an enhanced variability in number of seed capsules per plant and in sesame seed yield but decreased variability for plant height in mutant populations compared to the respective controls. Induced micro-mutations are responsible for the increase or decrease in the variability in these traits, and the segregation and recombination of polygenes in hybrid populations' results in cumulative effects. Similar findings have been noted elsewhere for seed yield, plant height, capsules, and seed yield [39][40][41]. A wide range of variability for different traits in F 2 M 2 populations has been observed by [19,42], who concluded that F 2 M 2 populations have the potential to offer a greater scope for evolving highyielding sesame varieties than either F 2 or M 2 populations.

Character correlation in the sesame F 2 M 2 generation
The phenotypic association between traits in genotypically diverse populations results from both genetic and environmental factors. The association between characters may alter in response to changes in a population's genotypic makeup. The relationship between characteristics may dissipate either completely or partially as a result of random, independent mutations in polygenes that affect several characteristics. For all populations of the F 2 M 2 generation (both untreated and treated), phenotypic and genotypic correlations among four characteristics were assessed and the results are shown in Tables 10 and 11.
The number of primary branches per sesame plant and the number of capsules per sesame plant both showed significant positive association with the number of seeds per sesame plant in all F 2 and F 2 M 2 populations at both phenotypic and genotypic levels. The number of capsules per plant and the seed yield per plant in sesame genotypes was positively correlated, which was also observed by [43][44][45][46][47]. A positive correlation between plant height, branch number, and capsule number per plant with sesame seed yield per plant was also observed by [46-better recombination of the genes controlling these traits are likely to have caused these association changes. Two cycles of mutagenic treatment resulted in increased variability, higher transgressive segregates, and improved mean and average transgression rates for sesame seed yield per plant compared to either the corresponding control or to one cycle of mutagenic treatment. These findings demonstrate the value of conducting two EMS treatment cycles to improve key characteristics in sesame: cumulative variation in hybrids is caused by segregation and artificial micromutations. Furthermore, the additionally variability created through the extended hybridization could be utilized for further breeding work, and the creation of even better seed yield segregants would be a further advance for selection in order to develop ever more superior varieties of sesame.